The determination of the quantity of DNA recovered from forensic samples as well as other samples is a critical step in the over all DNA typing process, but also in the detection of DNA in various other fields of science. A narrow range of input DNA from 0.5 to 2 ng is often needed to produce optimal results with for example multiplex DNA typing kits. Therefore, in order to ensure that a positive result is a positive result and/or a negative result is a negative result due to the absence of DNA, quantification of DNA is of absolute importance. Furthermore, the quality of standards for forensic DNA testing laboratories requires human-specific DNA quantification. This is due to isolation techniques that can recover human DNA as well as bacterial and other exogenous DNA. A number of procedures have been developed to permit quantification of human-specific DNA including start-blot techniques, liquid based hybridization assays and real-time PCR (polymerase chain reaction). Currently, real-time PCR is the dominant technique due to its wide dynamic range and ease of automation.
The modern STR-Kits have become much more sensitive and can obtain good results even using low amounts of DNA. Therefore, there is a desire for a method, kit and nucleic acid region that allows precise and accurate quantification of human DNA even in low concentrated samples. There are certain quantification and detection kits already available, however, these have serious drawbacks. One such kit is the Quantifiler Human Kit (Applied Biosystems) another kit is Quantifiler Duo Kit (Applied Biosystems) another kit is the Plexor HY Real-Time PCR Quantification Kit (Promega). Both the Quantifiler Duo Kit and the Plexor HY Kit target an autosomal and a gonosomal (Y-chromosome) target on the genome.
Drawbacks for the kits: According to LaSalle et al., (Forensic Science International: Genetics, “Analysis of Single and Multi-copy Methods for DNA Quantification by Real-Time Polymerase Chain Reaction”) the Quantifier Kits are more accurate in the quantification but have a lower dynamic range as the Plexor HY. The Plexor HY offers a higher dynamic range due to the amplification of a multi-copy target, but a lower accuracy. This lower accuracy can be attributed to the multicopy target. If less than the full set of 20 copies on a genome amplify, because of, for example, instability in the target copy number, than the ratio between the amplification between autosomal and gonosomal (Y) target may vary. The dynamic range of the Plexor HY kit is slightly better than that of the other kit (LaSalle et al., Forensic Science International: Genetics, “Analysis of Single and Multi-copy Methods for DNA Quantification by Real-Time Polymerase Chain Reaction”). In a statistical comparison LaSalle et al. demonstrated a significant difference between the two kits.
Another important parameter in forensics is the degradation grade of the DNA, that has to be analyzed. Since the amplicon size of the Quantifier Human and Plexor HY vary from 62 to 133 base pairs (bp), significant differences might be expected when the kits are applied to degraded DNA.
It should be emphasized that there are currently no official calibration standards or reference materials available especially for the quantification of proteins and nucleic acids (Ellison S L, English C A, Burns M J, Keer J T: Routes to improving the reliability of low level DNA analysis using real-time PCR. BMC Biotechnol 6: 33, 2006). A further problem arises from the pipetting error during the dilution of DNA quantification standards. In addition, long time stability of standards which is sometimes difficult in the case of natural polymers like nucleic acids and proteins must be guaranteed to achieve reproducibility. This is especially important in the case of low amounts of these substances. Furthermore, the manufacturing of reference substances has to be carried out under strict quality control to avoid secondary contaminations.
The Directive 2004/9/EC of THE EUROPEAN PARLIAMENT and of THE COUNCIL of 11 Feb. 2004 on the inspection and verification of good laboratory practice (GLP) (Official Journal of the European Union, Vol. L 50, pp. 28-43, 20 Feb. 2004) states in Part B (Revised guidance for the conduct of test facility inspections and study audits) following inspection and audit criteria for test systems and test and reference substances: “Test systems Purpose: to determine whether adequate procedures exist for the handling and control of the variety of test systems required by the studies undertaken in the facility, for example, chemical and physical systems, cellular and microbic systems, plants or animals ( . . . ). Test and reference substances Purpose: to determine whether the test facility has procedures designed (i) to ensure that the identity, potency, quantity and composition of test and reference substances are in accordance with their specifications, and (ii) to properly receive and store test and reference substances . . . ”.
According to DIN EN ISO 9000:2005 certified reference material (CRM) is a reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure that establishes metrological traceability to an accurate realization of the unit in which property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence.
Processing of the analytical results into DNA profiles also needs to be carefully controlled, and strict guidelines for the identification of alleles have to be developed and followed, to take account of such things as stutters, artefacts, peak size and morphology, variations in peak ratios, and so on. Three independent scientists, or two scientists and an expert system, are currently used in the UK to confirm the accurate designation of DNA profiles.
A centre piece is the quantification of template DNA. This has so far not been addressed adequately.